U.S. patent application number 16/980074 was filed with the patent office on 2021-01-14 for antibody specifically binding to folr1 and uses thereof.
This patent application is currently assigned to ALTEOGEN, INC.. The applicant listed for this patent is ALTEOGEN, INC.. Invention is credited to Hye-Shin CHUNG, Sunbae LEE, Soon Jae PARK.
Application Number | 20210009685 16/980074 |
Document ID | / |
Family ID | 1000005161660 |
Filed Date | 2021-01-14 |
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United States Patent
Application |
20210009685 |
Kind Code |
A1 |
PARK; Soon Jae ; et
al. |
January 14, 2021 |
ANTIBODY SPECIFICALLY BINDING TO FOLR1 AND USES THEREOF
Abstract
A modified antibody that binds specifically to folate receptor
alpha (FOLR1) and blocks the activity of FOLR1 with an increased
binding affinity, an antigen-binding fragment thereof, a
composition containing the antibody or fragment, and their uses are
disclosed. The modified antibody or antigen-binding fragment
thereof may be used for the prevention or treatment of cancer
proliferative disorder associated with an increased FOR1
expression, and may also be used for diagnosis of the disease. The
proliferative disorder may be cancer.
Inventors: |
PARK; Soon Jae; (Daejeon,
KR) ; CHUNG; Hye-Shin; (Daejeon, KR) ; LEE;
Sunbae; (Daejeon, KR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALTEOGEN, INC. |
Daejeon |
|
KR |
|
|
Assignee: |
ALTEOGEN, INC.
Daejeon
KR
|
Family ID: |
1000005161660 |
Appl. No.: |
16/980074 |
Filed: |
March 13, 2019 |
PCT Filed: |
March 13, 2019 |
PCT NO: |
PCT/KR2019/002911 |
371 Date: |
September 11, 2020 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 16/28 20130101;
A61K 47/6849 20170801; G01N 2333/705 20130101; C07K 2317/732
20130101; G01N 33/57492 20130101; C07K 2317/76 20130101; C07K
2317/92 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 47/68 20060101 A61K047/68; G01N 33/574 20060101
G01N033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 14, 2018 |
KR |
10-2018-0029762 |
Claims
1. A modified antibody or antigen-binding fragment thereof that
binds specifically to folate receptor-.alpha. (FOLR1).
2. The antibody or antigen-binding fragment thereof of claim 1,
wherein the antibody or antigen-binding fragment thereof inhibits
the biological activity of folate receptor-.alpha..
3. The antibody or antigen-binding fragment thereof of claim 1,
wherein the antibody or antigen-binding fragment thereof induces
antibody-dependent cellular cytotoxicity in cells that express
folate receptor-.alpha..
4. The antibody or antigen-binding fragment thereof of claim 1,
wherein the antibody or antigen-binding fragment thereof has a
dissociation constant of 1.times.10.sup.-7M or less for folate
receptor-.alpha..
5. The antibody or antigen-binding fragment thereof of claim 1,
which comprises: a heavy-chain CDR1 of SEQ ID NO: 3, a heavy-chain
CDR2 of SEQ ID NO: 4, and a heavy-chain CDR3 of SEQ ID NO: 5 or SEQ
ID NO: 28; and a light-chain CDR1 of SEQ ID NO: 6 or SEQ ID NO: 29,
a light-chain CDR2 of SEQ ID NO: 7 or SEQ ID NO: 30, and a
light-chain CDR3 of SEQ ID NO: 8.
6. The antibody or antigen-binding fragment thereof of claim 1,
which comprises: a heavy-chain variable region of SEQ ID NO: 1 or
SEQ ID NO: 31; and a light-chain variable region selected from the
group consisting of SEQ ID NO: 2 and SEQ ID NOs: 32 to 34.
7. The antibody or antigen-binding fragment thereof of claim 6,
which comprises: the heavy-chain variable region of SEQ ID NO: 1
and the light-chain variable region of SEQ ID NO: 32; or the
heavy-chain variable region of SEQ ID NO: 1 and the light-chain
variable region of SEQ ID NO: 33; or the heavy-chain variable
region of SEQ ID NO: 1 and the light-chain variable region of SEQ
ID NO: 34; or the heavy-chain variable region of SEQ ID NO: 31 and
the light-chain variable region of SEQ ID NO: 2; or the heavy-chain
variable region of SEQ ID NO: 31 and the light-chain variable
region of SEQ ID NO: 32.
8. An antibody-drug conjugate in which a drug is conjugated to the
antibody or antigen-binding fragment thereof of claim 1.
9. The antibody-drug conjugate of claim 8, wherein the antibody or
antigen-binding fragment thereof is conjugated to the drug via a
linker.
10. The antibody-drug conjugate of claim 8, wherein the drug is a
chemotherapeutic agent, a toxin, microRNA (miRNA), siRNA, shRNA, or
a radioactive isotope.
11. A composition comprising the antibody or antigen-binding
fragment thereof of claim 1; or the an antibody-drug conjugate
comprising a drug and the antibody or antigen-binding fragment
thereof wherein the drug is conjugated thereto.
12. (canceled)
13. A method of diagnosing a disorder associated with increased
expression of FOLR1, comprising contacting a test cell with the
antibody or antigen-binding fragment thereof of claim 1;
determining the level of expression of FOLR1 by the test cell by
detecting binding of the antibody or antigen-binding fragment
thereof to FOLR1; and comparing the level of expression of FOLR1 by
the test cell with the level of expression of FOLR1 by a control
cell, wherein a higher level of expression of FOLR1 by the test
cell as compared to the control cell indicates the presence of a
disorder associated with increased expression of FOLR1.
14. A method for preventing or treating cancer in a subject in need
thereof, comprising administering an effective amount of the
antibody or antigen-binding fragment thereof of claim 1; or an
antibody-drug conjugate comprising a drug and the antibody or
antigen-binding fragment thereof wherein the drug is conjugated
thereto, to the subject.
15. The method of claim 14, wherein the cancer is ovarian cancer,
breast cancer, lung cancer, kidney cancer, colon cancer, brain
cancer, rectal cancer, cervical cancer, or endometrial cancer.
16. The method of claim 13, wherein the test cell is obtained from
an individual suspected of having a disorder associated with
increased expression of FOLR1 and the disorder is a cell
proliferative disorder.
17. The composition of claim 11, which is a pharmaceutical
composition and further comprises a pharmaceutically acceptable
carrier.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a national stage entry application of
PCT/KR2019/002911 filed Mar. 13, 2019, which claims priority from
Korean Patent Application No. 10-2018-0029762 filed Mar. 14,
2018.
TECHNICAL FIELD
[0002] The present invention relates to an antibody that binds
specifically to folate receptor alpha (FOLR1) and blocks the
activity of FOLR1, the antibody being a modified antibody having
significantly increased binding affinity for an antigen compared to
that of the parent antibody. More particularly, the present
invention relates to an antibody or antigen-binding fragment
thereof that binds specifically to FOLR1, an antibody-drug
conjugate comprising the antibody or antigen-binding fragment
thereof, a pharmaceutical composition for preventing or treating
cancer comprising the same, and a composition for diagnosing
disease comprising the same.
BACKGROUND ART
[0003] Folate receptor alpha (FOLR1) is a protein which is
expressed at low to moderate levels in normal epithelial cells and
is overexpressed in certain epithelial-derived cancers such as
ovarian cancer, breast cancer, lung cancer, kidney cancer,
colorectal cancer, and endometrial cancer. In particular, FOLR1 is
overexpressed in more than 90% of ovarian cancer, and thus the
antibodies that target FOLR1 are useful for the treatment of cancer
(Sudimack and Lee, Adv. Drug Deliv. Rev. 2000, 41, 147-162). As a
representative example of a therapeutic antibody, farletuzumab
(MORAb-003) disclosed in US Patent Application Publication No.
2009/274697 (PCT International Publication No. 2005/080431) is a
humanized monoclonal antibody. Farletuzumab was developed by
Morphotek Inc., and has been reported as a potential therapeutic
agent for ovarian cancer. Farletuzumab is known to bind to FOLR1
with a binding affinity corresponding to a KD value of about 2 nM
(Grasso et al., Cancer Immun. 2007, 7, 6).
[0004] Typically, such therapeutic antibodies are extensively
engineered to possess desirable biological and physicochemical
properties, such as low immunogenicity, high affinity and
specificity, optimal effector functions, and good solubility and
stability. In particular, antibody humanization and affinity
maturation are the most frequently applied engineering processes
during the development of therapeutic antibody candidates. An
antibody humanization method is a method of replacing a
complementarity-determining region (CDR) of a non-human animal
antibody with a CDR of a human antibody. Humanized antibodies
resolve problems with non-human animal antibodies such as mouse
antibodies, such as high immunogenicity, low effector function, and
short blood half-life. By solving these problems, monoclonal
antibodies have been developed as pharmaceuticals, and various
humanized antibodies have already been approved for sale as
therapeutic antibodies. Although these humanized antibodies
actually show certain effects in clinical practice, it is also true
that their binding affinities for antigens are lower than those of
the original human antibodies, and that therapeutic antibodies
having higher effects are required. Since these problems may arise
due to the loss of affinity that results from direct grafting of
murine CDRs onto a human framework acceptor sequence, mutations in
CDRs or framework region (FR) residues supporting the structure of
CDR loops are often necessary.
[0005] In this respect, the application of antibody engineering
techniques to improve antibody efficacy is required. These
techniques include an affinity maturation technique of increasing
the affinity of an antibody for an antigen. Affinity maturation
refers to a technique of increasing the binding affinity of an
antibody for an antigen by introducing a random mutation into an
antibody gene, and may be very useful for the development of new
effective antibody drugs for therapeutic and diagnostic purposes.
For in vitro affinity maturation, three approaches are typically
used. These approaches include error-prone PCR, randomization of
targeted residues using degenerate oligonucleotides, and chain
shuffling. CDRs that may be selected as target residues are the
logical target for randomization because CDR-H3 and CDR-L3 tend to
dominate the antibody-antigen interaction. The binding affinity of
an antibody is increased by changing the amino acids in the CDR
region of the target antibody gene. It has been reported that,
through this method, the binding affinity of AKA (a humanized
antibody that binds to tumor-associated glycoprotein 72) was
increased 22-fold by changing the amino acids in CDR-H3 of AKA
(Hong et al., J. Biol. Chem. 2006, 281, 6985-6992), and the binding
affinity of a developed antibody for hepatitis B virus antigen was
also increased 6-fold (Hong el al., J. Microbiol. 2007, 45,
528-533).
[0006] A group of sequences having randomly arranged amino acids in
the CDR region may be referred to as a library. Since antibodies
coexist in the library, an operation of selecting antibodies from
the library is required. One of the most effective technologies of
selecting antibodies from the library is phage display technology.
This technology is based on a direct linkage between phage
phenotype and its encapsulated genotype, which leads to
presentation of molecule libraries on the phage surface. Phage
display is utilized in studying protein-ligand interactions and
receptor binding sites and in improving or modifying the affinity
of proteins for their binding partners.
[0007] Phage display involves the expression of selected proteins
on the surface of a filamentous phage through fusion with a phage
coat protein containing a genetic sequence that links a phenotype
to genotype selection. When combined with antibody libraries, phage
display allows for rapid in vitro selection of antigen-specific
antibodies and recovery of coding sequences corresponding thereto.
Large non-immune and synthetic human libraries have been
constructed as well as smaller immune libraries based on capturing
a single individual's immune repertoire. This completely in vitro
process allows for isolation of antibodies against poorly
immunogenic targets as well as those that cannot be obtained
through animal immunization, thus further expanding the utility of
the approach. Phage antibody display represents the first developed
methodology for high-throughput screening for human therapeutic
antibody candidates. Recently, other methods have been developed
for generation of fully human therapeutic antibodies, such as
single B-cell screening, next-generation genome sequencing and
transgenic mice with human embryonic stem cell with hepatitis B
genes. While each of these methods has particular advantages, phage
display has remained a key methodology for human antibody discovery
in terms of the ease and versatility of the screening method,
because it is a process that is performed in vitro. In addition,
panning, a method of selecting antibodies using phage display,
refers to a process that comprises immobilizing an antigen on an
immunotube and then adding an antibody library, displayed on the
phage surface, to the immunotube, and selecting only bound
antibodies through washing and elution processes. Phages carrying
Fabs bound or not bound to the antigen are isolated by repeated
washing. The antigen-bound phages are eluted off either through pH
change or protease digestion and re-infected into E. coli, from
which a new library enriched for antigen-binding clones can be
made. After this process is repeated several times, the library can
be sufficiently enriched so that the individual clones can be
isolated from E. coli stock (expressed as monoclonal phage), tested
and sequenced and the specific antibodies can be expressed.
[0008] Under this technical background, the present inventors have
recognized that there is an urgent need to develop an antibody
having excellent binding ability for FOLR1 to improve the efficacy
of an antibody against FOLR1, and have invented an antibody having
improved binding affinity for FOLR1 by introducing a mutation to
the complementarity-determining region. In addition, the present
inventors have constructed antibody libraries having amino acid
mutations induced in the CDRs of the heavy-chain and light-chain
variable regions of a parent antibody by affinity maturation, and
have selected individual antibodies having increased binding
affinity for FOLR1 by phage display technology, thereby completing
the present invention.
[0009] The above information disclosed in this Background section
is only for enhancement of understanding of the background of the
present invention. Therefore, it may not contain information that
forms a conventional art that is already known in the art to which
the present invention pertains.
SUMMARY
[0010] An object of the present invention is to provide an antibody
or antigen-binding fragment thereof that binds specifically to
FOLR1 and an antibody having further improved binding affinity for
an antigen compared to the above antibody.
[0011] Another object of the present invention is to provide an
antibody-drug conjugate in which a drug is conjugated to the
antibody or antigen-binding fragment thereof.
[0012] Still another object of the present invention is to provide
a pharmaceutical composition for preventing or treating cancer
comprising the antibody or antigen-binding fragment thereof or the
antibody-drug conjugate.
[0013] Yet another object of the present invention is to provide a
method for treating cancer comprising administering the antibody or
antigen-binding fragment thereof or the antibody-drug
conjugate.
[0014] Still yet another object of the present invention is to
provide the use of the antibody or antigen-binding fragment thereof
or the antibody-drug conjugate for treating cancer and the use of
the antibody or antigen-binding fragment thereof or the
antibody-drug conjugate in the manufacture of a medicament for
treating cancer.
[0015] A further object of the present invention is to provide a
composition for diagnosing disease comprising the antibody or
antigen-binding fragment thereof or the antibody-drug conjugate,
and a method for diagnosing disease using the antibody or
antigen-binding fragment thereof or the antibody-drug
conjugate.
[0016] To achieve the objects, an embodiment of the present
invention provides an antibody or antigen-binding fragment thereof
that binds specifically to folate receptor-.alpha. (FOLR1).
[0017] Preferably, the antibody or antigen-binding fragment thereof
may comprise six complementarity-determining regions (CDRs), and
the antibody or antigen-binding fragment thereof may comprise: a
heavy-chain CDR1 of SEQ ID NO: 3, a heavy-chain CDR2 of SEQ ID NO:
4, and a heavy-chain CDR3 of SEQ ID NO: 5 or SEQ ID NO: 28; and a
light-chain CDR1 of SEQ ID NO: 6 or SEQ ID NO: 29, a light-chain
CDR2 of SEQ ID NO: 7 or SEQ ID NO: 30, and a light-chain CDR3 of
SEQ ID NO: 8.
[0018] An embodiment of the present invention also provides an
antibody-drug conjugate comprising the antibody or antigen-binding
fragment thereof.
[0019] Another embodiment of the present invention also provides a
pharmaceutical composition for preventing or treating cancer, the
pharmaceutical composition comprising the antibody or
antigen-binding fragment thereof or the antibody-drug
conjugate.
[0020] Still another embodiment of the present invention also
provides a method for treating cancer, the method comprising
administering the antibody or antigen-binding fragment thereof or
the antibody-drug conjugate.
[0021] An embodiment of the present invention also provides the use
of the antibody or antigen-binding fragment thereof or the
antibody-drug conjugate for treating cancer and the use of the
antibody or antigen-binding fragment thereof or the antibody-drug
conjugate in the manufacture of a medicament for treating
cancer.
[0022] Another embodiment of the present invention also provides a
composition for diagnosing disease comprising the antibody or
antigen-binding fragment thereof or the antibody-drug conjugate,
and a method for diagnosing disease using the antibody or
antigen-binding fragment thereof or the antibody-drug
conjugate.
BRIEF DESCRIPTION OF DRAWINGS
[0023] FIG. 1 shows a comparison of the heavy-chain variable region
sequence of a parent antibody with that of a modified antibody. In
FIG. 1, matches between the sequences are marked with asterisks,
and one amino acid in the sequence of the CDR-H3 region is
different between the two antibodies.
[0024] FIG. 2 shows a comparison of the light-chain variable region
sequence of a parent antibody with that of a modified antibody. In
FIG. 2, matches between the sequences are marked with asterisks,
and the amino acid sequences of the CDR-L1 and CDR-L2 regions are
different between the two antibodies.
[0025] FIG. 3 shows the results of fractionating a modified
antibody by column purification using affinity resin
chromatography, and shows a chromatogram and the results of
SDS-PAGE analysis of each fraction (M: marker, LS: loading sample,
H: heavy chain, L: light chain, R: reducing condition).
[0026] FIG. 4 shows the results of ELISA that indicate the binding
affinity of each of a parent antibody and a modified antibody for
FOLR1 as a function of concentration.
[0027] FIG. 5 shows the results of SPR analysis performed to
compare the binding affinity of a parent antibody for FOLR1 with
the binding affinity of a modified antibody for FOLR1.
DETAILED DESCRIPTION AND PREFERRED EMBODIMENTS OF THE INVENTION
[0028] Unless otherwise defined, all technical and scientific terms
used in the present specification have the same meanings as
commonly understood by those skilled in the art to which the
present disclosure pertains. In general, the nomenclature used in
the present specification is well known and commonly used in the
art.
[0029] The term "antigen-binding fragment of an antibody" or
"antibody fragment" refers to a fragment having an antigen-binding
function, and includes Fab, F(ab'), F(ab')2 and Fv. Among antibody
fragments, Fab is a structure having light-chain and heavy-chain
variable regions, a light-chain constant region and a first
heavy-chain constant region (CH1), and has one antigen-binding
site. Fab' differs from Fab in that it has a hinge region
containing at least one cysteine residue at the C-terminus of the
heavy-chain CH1 region. An F(ab')2 antibody has a disulfide bond
formed by cysteine residues in the hinge region of Fab'. Fv is a
minimal antibody fragment having only a heavy-chain variable region
and a light-chain variable region, and recombinant techniques of
producing Fv fragments are disclosed in PCT International Patent
Publication Nos. WO 88/10649, WO 88/106630, WO 88/07085, WO
88/07086, and WO 88/09344.
[0030] The variable regions of the antibody used in the present
invention include three CDRs (CDR-H1, CDR-H2 and CDR-H3) in the
heavy-chain portion of the antibody and include three CDRs (CDR-L1,
CDR-L2 and CDR-L3) in the light-chain portion of the antibody.
These regions all form a loop and are regions that bind
specifically to an antigen.
[0031] In one aspect, the present invention is directed to an
antibody or antigen-binding fragment thereof that binds
specifically to folate receptor-.alpha. (FOLR1).
[0032] In an aspect of the present invention, the antibody or
antigen-binding fragment thereof may inhibit the biological
activity of folate receptor-.alpha.. Furthermore, the antibody or
antigen-binding fragment thereof may induce antibody-dependent
cellular cytotoxicity against cells that express folate
receptor-.alpha.. In addition, the antibody or antigen-binding
fragment thereof may have a dissociation constant of
1.times.10.sup.-7 M or less for folate receptor-.alpha..
[0033] As used herein, the term "parent antibody" refers to an
anti-FOLR1 antibody that binds specifically to FOLR1. In the
present invention, the antibody applied in a previous patent
application (US2005/0232919 A1) was used as the parent antibody.
The "parent antibody" in the present specification is one of the
antibodies specified in the previous patent application, and has a
heavy-chain sequence corresponding to SEQ ID NO: 1 below and a
light-chain sequence corresponding to SEQ ID NO: 2 below:
TABLE-US-00001 Heavy chain (SEQ ID NO: 1)
EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEWVAM
ISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVYFCARHG
DDPAWFAYWGQGTPVTVSS Light chain (SEQ ID NO: 2)
DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKPWIY
GTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSSYPYMYT FGQGTKVEIK
[0034] The scope of the present invention includes not only a
full-length antibody (full-length IgG) that binds specifically to
FOLR1, but also an antigen-binding fragment (fragmented IgG) of the
antibody molecule. The parent antibody used in the present
invention comprises CDRs represented by sequences of SEQ ID NOs: 3
to 8.
[0035] Therefore, in embodiments of the present invention, the
antibody or antigen-binding fragment thereof may comprise: the
heavy-chain CDR1 of SEQ ID NO: 3; the heavy-chain CDR2 of SEQ ID
NO: 4; the heavy-chain CDR3 of SEQ ID NO: 5; the light-chain CDR1
of SEQ ID NO: 6; the light-chain CDR2 of SEQ ID NO: 7; and the
light-chain CDR3 of SEQ ID NO: 8.
[0036] In an aspect of the present invention, a CDR library based
on the parent antibody was constructed as mentioned in PCT
International Patent Publication No. WO2016/114567DP, and a pComb3X
vector having the constructed library gene introduced therein is a
phagemid vector, which is a plasmid DNA having a phage origin of
replication and includes the phage surface protein pIII. The
library gene is ligated to the 5' end of the pIII gene and
expressed as a fusion protein in E. coli. VCSM13 helper phage is a
phage that provides necessary genetic information so that a
phagemid is assembled into a phage particle. The VCSM13 helper
phage contains the kanamycin antibiotic resistance gene so that E.
coli infected with the helper phage may be selected.
[0037] In addition, panning refers to a process of selectively
amplifying only clones, which bind to a specific molecule, from a
library of proteins, such as antibodies, displayed on the phage
surface. The procedure comprises: adding a phage library to a
target molecule immobilized on the surface to induce binding;
removing unbound phage clones by washing; eluting only bound phage
clones; re-infecting E. coli with the eluted phage clones; and
amplifying target-bound phage clones using helper phages. By
repeating this procedure, target-bound phage clones having a high
binding affinity for the target molecule immobilized on the surface
are selectively amplified.
[0038] As used herein, the term "modified antibody" refers to an
antibody made by modifying the parent antibody. The present
invention also provides a method for isolating and purifying the
modified antibody. A culture obtained by culturing under conditions
where the antibody protein is produced may be centrifuged to remove
impurities, and the resulting material may be purified using
affinity chromatography.
[0039] In addition, the modified antibody of an embodiment of the
present invention has binding affinity for FOLR1. The binding
affinity for FOLR1 may be measured using ELISA assay, SPR (surface
plasmon resonance) assay, or the like. Specifically, the binding
affinity may be measured by reacting an antibody composition with
FOLR1 immobilized on a plate at various concentrations,
additionally reacting a labeled antibody that recognizes the
antibody, and calculating the concentration of the antibody
composition bound to FOLR1. Thereby, it was possible to confirm
that the binding ability of the antibody obtained from multiple-CDR
libraries was improved more than that of the antibody obtained from
single-CDR libraries.
[0040] In an embodiment of the present invention, the modified
antibody may comprise the heavy-chain CDR3 of SEQ ID NO: 28, the
light-chain CDR1 of SEQ ID NO: 29, or the light-chain CDR2 of SEQ
ID NO: 30, which is a sequence modified from the parent
antibody.
[0041] Therefore, in an embodiment of the present invention, the
antibody or antigen-binding fragment thereof may comprise: the
heavy-chain CDR1 of SEQ ID NO: 3; the heavy-chain CDR2 of SEQ ID
NO: 4; the heavy-chain CDR3 of SEQ ID NO: 5 or SEQ ID NO: 28; the
light-chain CDR1 of SEQ ID NO: 6 or SEQ ID NO: 29; the light-chain
CDR2 of SEQ ID NO: 7 or SEQ ID NO: 30; and the light-chain CDR3 of
SEQ ID NO: 8.
[0042] In an aspect of the present invention, the antibody or
antigen-binding fragment thereof may comprise: a heavy-chain
variable region of SEQ ID NO: 1 or SEQ ID NO: 31; and a light-chain
variable region selected from the group consisting of SEQ ID NO: 2
and SEQ ID NOs: 32 to 34. Preferably, the antibody or
antigen-binding fragment thereof may comprise: the heavy-chain
variable region of SEQ ID NO: 1 and the light-chain variable region
of SEQ ID NO: 32; the heavy-chain variable region of SEQ ID NO: 1
and the light-chain variable region of SEQ ID NO: 33; the
heavy-chain variable region of SEQ ID NO: 1 and the light-chain
variable region of SEQ ID NO: 34; the heavy-chain variable region
of SEQ ID NO: 31 and the light-chain variable region of SEQ ID NO:
2; or the heavy-chain variable region of SEQ ID NO: 31 and the
light-chain variable region of SEQ ID NO: 32.
[0043] In another aspect, the present invention is directed to an
antibody-drug conjugate (ADC) in which a drug is conjugated to the
antibody or antigen-binding fragment thereof.
[0044] With regard to the antibody-drug conjugate (ADC), the
anticancer drug should remain stably bound to the antibody until
the anticancer drug is delivered to the target cancer cell. The
drug delivered to the target should be released from the antibody
and induce apoptosis of the target cell. To this end, the drug
should stably bind to the antibody, and at the same time, should
exhibit sufficient cytotoxicity to induce apoptosis of the target
cells when released in the target cell.
[0045] In an embodiment of the present invention, the antibody or
antigen-binding fragment thereof and a cytotoxic substance
comprising a drug such as an anticancer agent are bound to each
other (via, for example, a covalent bond, a peptide bond or the
like), and thus may be used as a conjugate or a fusion protein
(when a cytotoxic substance and/or labeling substance (marker) is
protein). The cytotoxic substance may be any substance which is
toxic to cancer cells, particularly solid cancer cells, and may be
at least one selected from the group consisting of radioisotopes,
cytotoxic compounds (small molecules), cytotoxic proteins, and
anticancer drugs, but is not limited thereto. The cytotoxic protein
may be at least one selected from the group consisting of ricin,
saporin, gelonin, momordin, deBouganin, diphtheria toxin,
pseudomonas toxin, and the like, but is not limited thereto. The
radioisotope may be at least one selected from the group consisting
of .sup.131I, .sup.188Rh and .sup.90Y, but is not limited thereto.
The cytotoxic compound may be at least one selected from the group
consisting of duocarmycin, monomethyl auristatin E (MMAE),
monomethyl auristatin F (MMAF),
N2'-diacetyl-N2'-(3-mercapto-1-oxopropyl)maytansine (DM1), PBD
(pyrrolobenzodiazepine) dimer, and the like, but is not limited
thereto.
[0046] In another embodiment of the present invention, the
antibody-drug conjugate may be obtained according to a method
well-known in the art.
[0047] In an aspect of the present invention, the antibody-drug
conjugate may be characterized in that the antibody or
antigen-binding fragment thereof is conjugated to the drug via a
linker.
[0048] In another aspect of the present invention, the linker may
be a cleavable linker or a non-cleavable linker.
[0049] The linker is a site for linking the antibody to the drug.
For example, the linker allows the drug to be released in a
cleavable form under an intracellular condition, that is, through
cleavage of the linker from the antibody in an intracellular
environment.
[0050] The linker may be a peptide linker that can be cleaved by a
cleavage agent present in an intracellular environment, for
example, in the lysosome or endosome, and can be cleaved by
intracellular peptidases or proteases, such as lysosome or endosome
proteases. Generally, a peptide linker is at least two amino acids
in length. The cleavage agent may include cathepsin B, cathepsin D
and plasmin, which hydrolyze the peptide to release the drug into
the target cell. The peptide linker can be cleaved by a
thiol-dependent protease cathepsin-B, which is highly expressed in
cancer tissue. For example, the peptide linker may be a Phe-Leu or
Gly-Phe-Leu-Gly linker. In addition, the peptide linker may, for
example, be a Val-Cit linker or a Phe-Lys linker, which can be
cleaved by an intracellular protease.
[0051] In an aspect of the present invention, the cleavable linker
is sensitive to pH and may be sensitive to hydrolysis at a certain
pH value. Generally, the pH-sensitive linker is a linker that can
be hydrolyzed under acidic conditions. Examples of acid-instable
linkers that can be hydrolyzed in lysosomes include hydrazone,
semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester,
acetal, ketal, and the like.
[0052] The linker may also be cleaved under reducing conditions,
and may, for example, be a disulfide linker. A variety of disulfide
bonds can be formed using N-succinimidyl-S-acetylthioacetate
(SATA), N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP),
N-succinimidyl-3-(2-pyridyldithio)butyrate (SPDB) and
N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene
(SMPT).
[0053] In an embodiment of the present invention, the drug and/or
the drug-linker may be randomly conjugated through the lysine of
the antibody, or may be conjugated through cysteine, which is
exposed when the disulfide bond chain is reduced. In some cases,
the linker-drug can be conjugated through cysteine present in a
genetically engineered tag, e.g., a peptide or protein. The
genetically engineered tag, e.g., a peptide or protein, may include
an amino acid motif that can be recognized, for example, by an
isoprenoid transferase. The peptide or protein has a deletion at
the carboxyl terminus of the peptide or protein or an addition at
the carboxyl (C) terminus of the peptide or protein through a
covalent bond of a spacer unit. The peptide or protein may be
covalently bonded directly to an amino acid motif, or may be linked
to the amino acid motif through a covalent bond to the spacer unit.
The amino acid spacer unit consists of 1 to 20 amino acids, and is
particularly preferably a glycine unit.
[0054] The linker may comprise a beta-glucuronide linker that is
recognized and hydrolyzed by beta-glucuronidase, which is present
in multiple copies in the lysosome or overexpressed in some tumor
cells. Unlike the peptide linker, this linker has the advantage of
increasing the solubility of the antibody-drug conjugate when bound
to a drug having high hydrophobicity due to the high hydrophilicity
thereof.
[0055] In this regard, in embodiments of the present invention, it
is possible to use a beta-glucuronide linker disclosed in Korean
Patent Application Publication No. 2015-0137015, for example, a
beta-glucuronide linker including a self-immolative group.
[0056] In addition, the linker may be, for example, a non-cleavable
linker, and the drug may be released merely through a single step
of hydrolyzing the antibody, thus producing, for example, an amino
acid/linker/drug complex. This type of linker can be a thioether
group or a maleimidocaproyl group, and is stable in the blood.
[0057] In an aspect of the present invention, the drug may be a
chemotherapeutic agent, a toxin, microRNA (miRNA), siRNA, shRNA, or
a radioactive isotope. The drug, which is an agent having a
pharmacological effect, may be conjugated to the antibody.
[0058] The chemotherapeutic agent may be a cytotoxic agent or an
immunosuppressive agent. Specifically, the chemotherapeutic agent
may comprise a microtubulin inhibitor, a mitotic inhibitor, a
topoisomerase inhibitor, or a chemotherapeutic agent capable of
functioning as a DNA intercalator. The chemotherapeutic agent may
also comprise an immunomodulatory compound, an anticancer agent, an
antiviral agent, an antibacterial agent, an antifungal agent, an
anthelmintic, or a combination thereof.
[0059] For example, the drug may comprise at least one selected
from the group consisting of maytansinoid, auristatin, aminopterin,
actinomycin, bleomycin, thalidomide, camptothecin,
N8-acetylspermidine, 1-(2 chloroethyl)-1,2-dimethyl sulfonyl
hydrazide, esperamycin, etoposide, 6-mercaptopurine, dolastatin,
trichothecene, calicheamicin, taxol, taxane, paclitaxel, docetaxel,
methotrexate, vincristine, vinblastine, doxorubicin, melphalan,
chlorambucil, duocarmycin, L-asparaginase, mercaptopurine,
thioguanine, hydroxyurea, cytarabine, cyclophosphamide, ifosfamide,
nitrosourea, cisplatin, carboplatin, mitomycins (mitomycin A and
mitomycin C), dacarbazine, procarbazine, topotecan, nitrogen
mustard, cytoxan, etoposide, 5-fluorouracil, CNU
(bis-chloroethylnitrosourea), irinotecan, camptothecin, bleomycin,
idarubicin, daunorubicin, dactinomycin, plicamycin, asparaginase,
vinorelbine, chlorambucil, melphalan, carmustine, lomustine,
busulfan, treosulfan, dacarbazine, etoposide, teniposide,
topotecan, 9-aminocamptothecin, crisnatol, trimetrexate,
mycophenolic acid, tiazofurin, ribavirin, EICAR
(5-ethynyl-1-beta-ribofuranosylimidazole-4-carboxamide),
hydroxyurea, deferoxamine, floxuridine, doxifluridine, raltitrexed,
cytarabine (ara C), cytosine arabinoside, fludarabine, tamoxifen,
raloxifene, megestrol, goserelin, leuprolide acetate, flutamide,
bicalutamide, EB1089, CB1093, KH1060, verteporfin, phthalocyanine,
photosensitizer Pe4, demethoxy-hypocrellin A, interferon-.alpha.,
interferon-.gamma., tumor necrosis factor, gemcitabine, Velcade,
Revlimid, Thalomid, lovastatin, 1-methyl-4-phenylpyridiniumion,
staurosporine, actinomycin D, dactinomycin, bleomycin A2, bleomycin
B2, peplomycin, epirubicin, pirarubicin, zorubicin, mitoxantrone,
verapamil and thapsigargin, nucleases, and toxins derived from
bacteria or plants and animals, but the present invention is not
limited thereto.
[0060] In an aspect of the present invention, the drug may have a
nucleophile group selected from the group consisting of amine,
thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone,
hydrazine carboxylate and aryl hydrazide groups, which can react
with an electrophilic group on the linker and the linker reagent to
form a covalent bond.
[0061] In still another aspect, the present invention is directed
to a pharmaceutical composition for preventing and/or treating
cancer, the pharmaceutical composition comprising the antibody or
antigen-binding fragment thereof or the antibody-drug
conjugate.
[0062] In yet another aspect, the present invention is directed to
a method for treating cancer, the method comprising administering
the antibody or antigen-binding fragment thereof or the
antibody-drug conjugate to a patient in need of prevention or
treatment.
[0063] In still yet another aspect, the present invention is
directed to the use of the antibody or antigen-binding fragment
thereof or the antibody-drug conjugate for treating cancer.
[0064] In further another aspect, the present invention is directed
to the use of the antibody or antigen-binding fragment thereof or
the antibody-drug conjugate in the manufacture of a medicament for
treating cancer.
[0065] In an aspect of the present invention, the cancer may be
ovarian cancer, breast cancer, lung cancer, kidney cancer, colon
cancer, brain cancer, rectal cancer, cervical cancer, or
endometrial cancer, but is not limited thereto.
[0066] Although the pharmaceutical composition comprising the
antibody or antigen-binding fragment thereof or the antibody-drug
conjugate according to embodiments of the present invention may
also comprise only the antibody or antigen-binding fragment thereof
or the antibody-drug conjugate as an active ingredient, it is
generally mixed with one or more pharmacologically acceptable
carriers, and is preferably provided as a pharmaceutical
formulation prepared by any method known in the technical field of
pharmaceuticals.
[0067] The pharmaceutical composition of embodiments of the present
invention may be used alone or in combination with at least one
therapeutic drug selected from the above-described radioisotopes,
low-molecular-weight drugs, polymer drugs or antibody drugs. In
addition, the pharmaceutical composition of embodiments of the
present invention may be used in combination with a conventional
therapeutic agent. That is, the pharmaceutical composition
comprising the antibody or antigen-binding fragment thereof or the
antibody-drug conjugate according to an embodiment the present
invention may be administered simultaneously or sequentially with a
conventional therapeutic agent such as an anticancer agent.
[0068] As the route of administration, it is preferable to use the
most effective route of administration at the time of treatment.
Examples of the route of administration include oral administration
or parenteral administration such as intra-mouth, intra-airway,
intrarectal, subcutaneous, intramuscular or intravenous
administration. Intravenous administration is preferred.
[0069] Dosage forms include a spray, a capsule, a tablet, a powder,
a granule, a syrup, an emulsion, a suppository, an injection, an
ointment, or a tape.
[0070] The dosage or the frequency of administration varies
according to the desired therapeutic effect, the mode of
administration, the period of treatment, and the patient's age and
body weight, but is usually 10 .mu.g/kg to 10 mg/kg per day for an
adult.
[0071] Since the antibody or antigen-binding fragment thereof
according to the present invention binds specifically to folate
receptor-.alpha. (FOLR1), FOLR1 may be detected or diagnosed using
the same. Expression of FOLR1 is related to several diseases, for
example, cancer.
[0072] Therefore, in another aspect, the present disclosure is
directed to a composition for diagnosing disease, the composition
comprising the antibody or antigen-binding fragment thereof.
[0073] In an aspect of the present invention, the disease may be
FOLR1-related disease, for example, cancer, but is not limited
thereto.
[0074] In still another aspect, the present invention is directed
to a method for diagnosing disease or a method for providing
information for diagnosing disease, the method comprising a step of
treating (administering) a biological sample isolated from a
subject with the antibody or antigen-binding fragment thereof.
[0075] In another aspect of the present invention, the method for
diagnosing disease may further comprise, after the treatment step,
a step of identifying whether an antigen-antibody reaction occurs.
In the detection method, when the antigen-antibody reaction is
detected, a FOLR1-related disease, for example, cancer, may be
determined to be present in the biological sample or a patient from
which the biological sample has been obtained. Thus, the method may
further comprise, after the step of identifying, a step of
determining that, when the antigen-antibody reaction is detected,
the biological sample or the patient is a FOLR1-related disease
patient, for example, a cancer patient. The biological sample may
be selected from the group consisting of cells, tissues, body
fluids, cultures thereof and the like, obtained (isolated) from a
mammal such as a human (e.g., a cancer patient).
[0076] The step of identifying whether or not the antigen-antibody
reaction occurs may be performed through various methods known in
the art. For example, the step may be performed through a
conventional enzymatic reaction, fluorescence, luminescence and/or
radiation detection. Specifically, the step may be performed by a
method selected from the group consisting of immunochromatography,
immunohistochemistry, enzyme-linked immunosorbent assay (ELISA),
radioimmunoassay (RIA), enzyme immunoassay (EIA), fluorescence
immunoassay (FIA), luminescence immunoassay (LIA), Western
blotting, microarray, and immunoprecipitation assay, but is not
limited thereto.
[0077] In this case, the antibody or antigen-binding fragment
thereof may further comprise a marker. The marker may be at least
one selected from the group consisting of radioactive isotopes,
fluorescent substances, chromogen and dyeing substances. The marker
may be bound (linked) to the antibody or antigen-binding fragment
by a conventional method (for example, a chemical bond such as a
covalent bond, coordination bond or ionic bond). The binding of the
antibody (or antigen-binding fragment) to the marker may be
performed in accordance with techniques known in the art.
[0078] Hereinafter, various embodiments of the present invention
will be described in more detail with reference to examples. It
will be obvious to those skilled in the art that these examples are
merely to illustrate the present invention, and the scope of the
present invention is not limited by these examples.
Example 1: Selection of Library Clones
[0079] To obtain optimum sequences having improved binding affinity
for FOLR1, CDR libraries were constructed using Fab fragments.
Example 1-1: Preparation of Parent Antibody Fab Template
[0080] Variable regions were synthesized from the light chain and
heavy chain of a parent antibody, respectively, and constant
regions were synthesized from pComb3X-TT. PCR reaction was
performed under the following conditions: pre-denaturation at
94.degree. C. for 2 min, and then 25 cycles, each consisting of 30
sec at 94.degree. C., 30 sec at 56.degree. C. and 30 sec at
72.degree. C., followed by elongation at 72.degree. C. for 7 min.
In the PCR reaction, 100 ng of one template was used, or a mixture
obtained by mixing two templates in an amount of 3 .mu.L of each
template was used. 3 .mu.L of each primer was used at a
concentration of 20 pM, 0.05 mM dNTP and 0.5 .mu.L (2.6 units) Taq
polymerase were used, and the reaction volume was 100 .mu.L. After
completion of the reaction, whether amplification occurred was
checked using 1% agarose gel electrophoresis, and the amplification
product was purified using a QIAGEN' gel extraction kit. For second
and third PCR, overlapping PCR was performed using the amplified
fragment as a template. The PCR reaction product DNA was purified
on an agarose gel using a Qiagen gel extraction kit, cleaved with a
SfiI restriction enzyme, and then subjected to gel extraction. 153
ng of the SfiI-cleaved antibody gene and 136 ng of the SfiI-cleaved
pComb3X vector were mixed with each other, added to 10.times.T4 DNA
ligation buffer and 10 units of ligase, reacted at room temperature
for 3 hours, and then heat-shocked at 42.degree. C. for 45 seconds
in E. coli DH5a cells, followed by incubation at 37.degree. C. for
1.5 hours. From the colony obtained by the above-described
transformation method, a template for antibody library construction
composed of a 50-kDa Fab fragment was obtained.
Example 1-2: Antibody Library Construction
[0081] Libraries were constructed by artificially introducing
diversity into the complementarity-determining region, and the CDRs
and FRs of a given antibody can be identified according to the
procedure of UCL, Antibody Informatics
(www.bioinf.org.uk/abs/).
[0082] Libraries were constructed by randomizing the CDR sequences
of the parent antibody based on the template prepared in Example
1-1. Among the six CDRs of the parent antibody, CDR-H2 was excluded
from the experiment because it was so long as to be difficult to
handle in the experiment. The CDR sequence of the parent antibody
is shown in Table 1 below.
TABLE-US-00002 TABLE 1 CDR sequence of parent antibody Residue
Residue located located CDR in front Sequence at back CDR-
Cys-Xaa-Xaa-Xaa GFTFSGYGLS Trp-Val H1 (SEQ ID NO: 51) (SEQ ID NO:
3) CDR- Leu-Gln-Trp-Val- MISSGGSYTYYADSV Lys H2 Ala (SEQ ID NO: 4)
(SEQ ID NO: 52) CDR- Cys-Ala-Arg HGDDPAWFAY Trp-Gly-Gln- H3 (SEQ ID
NO: 5) Gly (SEQ ID NO: 9) CDR- Cys SVSSSISSNNLH Trp L1 (SEQ ID NO:
6) CDR- Ile-Tyr GTSNLAS Gly L2 (SEQ ID NO: 7) CDR- Cys QQWSSYPYMYT
Phe-Gly-Gln- L3 (SEQ ID NO: 8) Gly (SEQ ID NO: 54)
[0083] To randomize a specific position in the region that binds to
an antigen, primers were prepared using mixed base codes (Table 2).
The mixed base code is a degenerated primer and refers to an
oligonucleotide in which two or more bases exist in one position so
that they can bind to similar nucleotide sequences in consideration
of the nucleotide sequence similarity. Prior to preparation of the
primers, in order to determine the specific position to be
randomized, conserved residues were identified through CDR sequence
analysis of the parent antibody. For codon diversification of a
portion excluding these residues, primers were prepared using mixed
base codes.
TABLE-US-00003 TABLE 2 Primer sequences SEQ ID CDR Primer Sequence
(5' .fwdarw. 3') NO: CDR- Farl-H1- GCC TCT GGC TTC ACT TTC AGT RRT
TAC 9 H1 random-f GVT MTG ART TGG GTG AGA CAG GCA CCT G Fanl-H1-b
ACT GAA AGT GAA GCC AGA GGC 10 CDR- Farl-H3- GGG GTC TAT TTT TGT
GCA AGA NNK NNK 11 H3 random1-f GAC GAT CCA GCA TGG TTT GMT TAC TGG
GGC CAA GGG ACC Farl-H3- GGG GTC TAT TTT TGT GCA AGA CAC NNK 12
random2-f NNK GAT CCA GCA TGG TTT GMT TAC TGG GGC CAA GGG ACC
Farl-H3- GGG GTC TAT TTT TGT GCA AGA CAC GGT 13 random3-f NNK NNK
CCA GCA TGG TTT GMT TAC TGG GGC CAA GGG ACC Farl-H3- GGG GTC TAT
TTT TGT GCA AGA CAC GGT 14 random4-f GAC NNK NNK GCA TGG TTT GMT
TAC TGG GGC CAA GGG ACC Farl-H3- GGG GTC TAT TTT TGT GCA AGA CAC
GGT 15 random5-f GAC GAT NNK NNK TGG TTT GMT TAC TGG GGC CAA GGG
ACC Farl-H3- GGG GTC TAT TTT TGT GCA AGA CAC GGT 16 random6-f GAC
GAT CCA NNK NNK TTT GMT TAC TGG GGC CAA GGG ACC Farl-H3-b TCT TGC
ACA AAA ATA GAC CCC 17 CDR- Farl-L1- AC AGA GTC ACC ATC ACA TGC AGK
GYT 18 L1 random-f TCC TCC RGT VTT AGT TCA ARC WAT CTG MAC TGG TAT
CAG CAG AAG CCC G Farl-L1-b GCA TGT GAT GGT GAC TCT GT 19 CDR-
Farl-L2- C CCA AAG CCC TGG ATC TAC GVT RCC TCT 20 L2 random-f AVT
CKG GMA AST GGG GTG CCT TCA AGG TTC A Farl-L2-b GTA GAT CCA GGG CTT
TGG G 21 CDR- Farl-L3- GCA ACT TAC TAT TGC CAG CAG NNK BMT 22 L3
random-f WAT TWT CCA YMT NNK YMC ACC TTC GGT CAG GGC AC Farl-L3-b
CTG CTG GCA ATA GTA AGT TGC 23 All pC3X-f GCA CGA CAG GTT TCC CGAC
24 CDRs pC3X-b AAC CAT CGA TAG CAG CAC CG 25 CDR- Lead-b GGC CAT
GGC TGG TTG GGC 26 L3H3 Lead-VH GCC CAA CCA GCC ATG GCC 27
[0084] For codon randomization of the parent antibody CDR-L1, L2,
L3, H1 and H2 regions, each of the regions was amplified by PCR and
attached to PCR-amplified CDR DNA through overlap extension PCR,
thereby constructing single-CDR libraries having diversity only in
one CDR and multiple-CDR libraries having diversity in two CDRs.
The PCR reaction was performed under the following conditions:
pre-denaturation at 94.degree. C. for 2 min and then 25 cycles,
each consisting of 30 sec at 94.degree. C., 30 sec at 56.degree. C.
and 30 sec at 72.degree. C., followed by extension at 72.degree. C.
for 7 min. However, the elongation time at 72.degree. C. was
adjusted to 1 min 30 sec or 2 min depending on the length of
predicted DNA fragment products. Specifically, the elongation time
was 1 min 30 sec for a predicted length of 500 to 1,500 bp and 2
min for a predicted length of 1,500 to 2,000 bp. The names of the
templates, primers and products used in these processes are
summarized in Table 3 below. The constructed single- or
multiple-CDR library was isolated and purified by 1% agarose gel
electrophoresis, and the purification product was cleaved by
treatment with a SfiI restriction enzyme at 50.degree. C. for 12
hours or more. The pComb3X phagemid vector was also cleaved with a
SfiI restriction enzyme in the same manner.
TABLE-US-00004 TABLE 3 PCR templates and primers for library
construction Primer SEQ ID PCR Template NOS: Product Single-CDR
library CDR-L1 1.sup.st pFAR-FabC3 19, 24 FAR-L1-F 18, 25 FAR-L1-R
2.sup.nd FAR-L1-F, FAR-L1-R 24, 25 FAR-CDR-L1 CDR-L2 1.sup.st
pFAR-FabC3 21, 24 FAR-L2-F 20, 24 FAR-L2-R 2.sup.nd FAR-L2-F,
FAR-L2-R 24, 25 FAR-CDR-L2 CDR-L3 1.sup.st pFAR-FabC3 23, 24
FAR-L3-F 22, 25 FAR-L3-R 2.sup.nd FAR-L3-F, FAR-L3-R 24, 25
FAR-CDR-L3 CDR-H1 1.sup.st pFAR-FabC3 10, 24 FAR-H1-F 9, 25
FAR-H1-R 2.sup.nd FAR-H1-F, FAR-H1-R 24, 25 FAR-CDR-H1 CDR-H3
1.sup.st pFAR-FabC3 17, 24 FAR-H3-F 11 to FAR-H3-R 16, 25 2.sup.nd
FAR-H3-F, FAR-H3-R 24, 25 FAR-CDR-H3 Multiple- CDR library CDR-L3H3
1.sup.st pFAR CDR-L3 24, 26 FAR-L3 pFAR-CDR-H3 25, 27 FAR-H3
2.sup.nd FAR-L3, FAR-H3 24, 25 FAR-CDR-L3H3
[0085] For ligation, the SfiI-cleaved vector and the SfiI-cleaved
insert were mixed together in equal amounts and reacted overnight
at room temperature. If the volume of the ligation product is too
large for transformation, the volume of the ligation product can be
reduced using EtOH precipitation, and the method for reducing the
volume is as follows. To 50 .mu.L of the ligation product, 5 .mu.L
(1/10) of 3 M sodium acetate (pH 5.2) and 110 .mu.L (2-fold) of
100% EtOH were added, and DNA was allowed to precipitate at
-20.degree. C. for 2 hours or more. The precipitated DNA was
centrifuged at 12,000 rpm for 15 minutes, and then washed with 1 ml
of 70% EtOH and centrifuged under the same conditions. The pellet
was dried, and then dissolved in 10 .mu.L of deionized water.
Transformation was performed by electroporation. Specifically, 10
.mu.L of the ligation product and 50 .mu.L of E. coli TG1 competent
cells were mixed together and then placed in a 0.2-cm cooled
cuvette, which was placed in an electroporator. Next, the cells
were pulsed at 2.5 kV for 4 to 5 msec. 2 mL of recovery medium
heated to 37.degree. C. was added thereto, immediately after
pulsing and then incubation was performed at 37.degree. C. for 1
hour. Next, 1 .mu.L of the incubated cells was diluted 1,000-fold
with SB medium, and 10 .mu.L and 100 .mu.L of the cell dilution
were dispensed on an LB agar plate to prepare samples for measuring
the library size, and the remainder was plated on one plate and
incubated overnight at 37.degree. C. The next day, the library size
was measured by counting the number of colonies on the plate into
which the diluted cells were dispensed. In addition, 5 mL of SB
medium was added to the plate into which the undiluted cells were
dispensed, the cells were collected using a spreader, and then a
0.5-fold volume of 50% glycerol was added to the cells, which were
then stored in a deep freezer (-75.degree. C.)
TABLE-US-00005 TABLE 4 CDR library Library size CDR-L1 4.56 .times.
10.sup.6 CDR-L2 2.94 .times. 10.sup.7 CDR-L3 6.22 .times. 10.sup.7
CDR-H1 4.78 .times. 10.sup.6 CDR-H3 1.30 .times. 10.sup.8 CDR-L3H3
8.82 .times. 10.sup.7
[0086] The size of the obtained library indicates transformation
efficiency, specifically the number of individual clones. As a
result, it can be considered that antigen-binding diversity
corresponds to the library size. That is, the following libraries
were prepared: a CDR-L1 library having a diversity of 10.sup.6, a
CDR-L2 library having a diversity of 10.sup.7, a CDR-L3 library
having a diversity of 10.sup.7, a CDR-H1 library having a diversity
of 10.sup.6, a CDR-H3 library having a diversity of 10.sup.8, and a
CDR-L3H3 library having a diversity of 10.sup.7 (Table 4).
Example 1-3: Selection by ELISA after Phage-Display Panning
[0087] Panning was performed to select a library clone that binds
to human FOLR1, and as panning was repeated, a clone having further
increased binding affinity for FOLR1 could be obtained.
[0088] For library amplification and recovery of a Fab-expressing
bacteriophage, 100 .mu.L of a TG1 stock transformed with the
library was seeded into 20 mL of SB/Amp+2% glucose medium, and the
library was expressed in E. coli TG1 cells at 37.degree. C. and 220
rpm for 1.5 to 2 hours. The cell culture was centrifuged at 3500
rpm for 15 minutes. The supernatant was removed, and the pellet was
re-suspended in 20 ml of SB/Amp medium. 0.5 mL of helper phage
VCSM13 (about 1011 pfu) was added to the suspension, which was then
infected with the helper phage by culture at 37.degree. C. at 120
rpm for 1 hour. Next, kanamycin (50 mg/mL) was added to the
suspension to reach 70 .mu.g/mL, followed by culture at 30.degree.
C. at 200 rpm for 16 hours. The culture was centrifuged, and 5 mL
of 5.times.PEG concentrated solution was added to the
phage-containing supernatant, which was then concentrated on ice
for 30 minutes. The concentrate was centrifuged at 12,000 rpm for
15 minutes, and the supernatant was removed. The phage pellet was
re-suspended in 0.3 mL of PBS to obtain a library phage (for
storage, a 0.5-fold volume of 50% glycerol is added to the library
phage which is then stored at -75.degree. C.)
[0089] To amplify the obtained library phage, an E. coli TG1 cell
stock was seeded into 10 mL of SB medium, and cultured in an
incubator at 37.degree. C. at 220 rpm for about 4 to 5 hours up to
the mid-log phase (OD.sub.600=0.5 to 1.0), thus preparing competent
cells which were then stored at 4.degree. C. until use. Panning was
performed in the following manner. 1 .mu.g/mL of FOLR1 was coated
on an immunotube, and then blocked with a blocking solution (3%
skim milk) at 37.degree. C. for 1 hour. 0.5 mL of the library phage
was blocked by adding 0.5 mL of blocking solution thereto at a
ratio of 1:1, and then allowed to react with the immobilized
antigen. After the reaction had proceeded for one hour or more,
unreacted or weakly bound phage was removed through a washing step
with PBS-Tween20 buffer, and strongly bound phage was eluted out
with 1 mL of TEA (100 mM) for 10 minutes and then neutralized with
0.5 mL of Tris-HCl (1 M, pH 7.4). 1.5 mL of the eluted phage was
added to 8.5 mL of the competent cells and infected into the cells
in an incubator at 37.degree. C. at 120 rpm for 1 hour. Next, to
measure the library size, 1 .mu.L of the 2 mL reaction solution was
diluted 1,000-fold and 10,000-fold and plated, and the remaining
reaction solution was plated on one plate and incubated overnight
at 37.degree. C.
[0090] The transformed E. coli library was cultured and infected
with VCSM13 helper phage to obtain an antibody phage library having
Fab clones displayed on the surface thereof. Only phage clones that
bound strongly to FOLR1 were selected by panning the library for 3
to 5 rounds against FOLR1 adsorbed on the immune-tube surface.
Through this process, clones that bound weakly to FOLR1 or did not
bind to FOLR1 due to defects in the synthesized CDR sequence were
removed, and as a result, it was possible to select CDR sequences
that had no defects and were better optimized than the existing
sequences. The CDR-L1 and CDR-L2 libraries were panned for 5
rounds, the CDR-L3, CDR-H3 and CDR-L3H3 libraries were panned for 4
rounds, and the CDR-H1 library was panned for 3 rounds. For each
panning round, the ratio (0/I ratio) of the eluted phage (output
phage) to the phage (input phage) used in panning was calculated,
and the results were expressed as % bound in Table 5 below. The
fact that similar % bound values appear even when panning is
repeated indicates that the binding affinity for FOLR1 reached
saturation (Table 5). When this result appeared, panning was no
longer performed, and the next step was performed using the eluted
phage. To validate the functionality of the antibody phage
libraries that resulted from panning, 94 clones were screened from
each CDR library by ELISA. The number of ELISA-positive clones
showing a binding signal at least 3 times stronger than the
background signal was identified to be 92 for CDR-L1, 66 for
CDR-L2, 19 for CDR-L3, 94 for CDR-H1, 48 for CDR-H3, and 63 for
CDR-L3H3. Eight clones among the clones showing a stronger binding
signal for each library were sequenced (Table 6).
TABLE-US-00006 TABLE 5 Panning conditions and results Number Pan-
Phage Phage of Amount Phage ning input output % wash- of library
round (c.f.u) (c.f.u) Bound ings antigen CDR- 1 .sup. 3.6 .times.
10.sup.10 5.1 .times. 10.sup.8 1.4 3 1.0 .mu.g L1 2 ~10.sup.10 8.5
.times. 10.sup.8 8.5 5 0.5 .mu.g 3 ~10.sup.10 1.1 .times. 10.sup.9
11 10 0.1 .mu.g 4 ~10.sup.10 5.9 .times. 10.sup.8 5.9 10 0.1 .mu.g
5 ~10.sup.10 4.3 .times. 10.sup.8 4.3 10 0.1 .mu.g CDR- 1 .sup. 2.5
.times. 10.sup.10 6.8 .times. 10.sup.8 2.7 3 1.0 .mu.g L2 2
~10.sup.10 7.5 .times. 10.sup.8 7.5 5 0.5 .mu.g 3 ~10.sup.10 8.0
.times. 10.sup.8 8.0 10 0.1 .mu.g 4 ~10.sup.10 3.2 .times. 10.sup.8
3.2 10 0.1 .mu.g 5 ~10.sup.10 3.1 .times. 10.sup.8 3.1 10 0.1 .mu.g
CDR- 1 1.4 .times. 10.sup.9 4.3 .times. 10.sup.6 0.3 3 1.0 .mu.g L3
2 3.7 .times. 10.sup.8 1.0 .times. 10.sup.5 2.7 .times. 10.sup.-2 5
0.5 .mu.g 3 1.7 .times. 10.sup.9 7.2 .times. 10.sup.7 4.2 5 0.5
.mu.g 4 5.8 .times. 10.sup.9 8.0 .times. 10.sup.7 1.4 10 0.1 .mu.g
CDR- 1 .sup. 5.5 .times. 10.sup.10 4.9 .times. 10.sup.8 0.9 3 1.0
.mu.g H1 2 ~10.sup.10 6.2 .times. 10.sup.8 6.2 5 0.5 .mu.g 3
~10.sup.10 6.5 .times. 10.sup.8 6.5 10 0.1 .mu.g CDR- 1 1.9 .times.
10.sup.9 2.2 .times. 10.sup.7 1.2 3 1.0 .mu.g H3 2 3.5 .times.
10.sup.8 1.0 .times. 10.sup.5 2.9 .times. 10.sup.-2 5 0.5 .mu.g 3
1.8 .times. 10.sup.9 8.6 .times. 10.sup.7 4.8 5 0.5 .mu.g 4 4.4
.times. 10.sup.9 1.1 .times. 10.sup.8 2.5 10 0.1 .mu.g CDR- 1 8.9
.times. 10.sup.8 1.0 .times. 10.sup.8 11.2 3 1.0 .mu.g L3H3 2 6.0
.times. 10.sup.8 1.0 .times. 10.sup.5 1.7 .times. 10.sup.-2 5 0.5
.mu.g 3 1.8 .times. 10.sup.9 2.6 .times. 10.sup.7 1.4 5 0.5 .mu.g 4
3.2 .times. 10.sup.9 4.0 .times. 10.sup.7 1.3 10 0.1 .mu.g
TABLE-US-00007 TABLE 6 Sequencing results CDR Clone Sequencing
results (SEQ ID NO:) CDR- WT SVSSSISSNNLH (SEQ ID NO: 6) L1 C6, E6
SASSGLSSSYLH (SEQ ID NO: 29) C7 SASSSLSSSYLH (SEQ ID NO: 35) D5
RVSSGISSNNLH (SEQ ID NO: 36) D7 RASSGLSSNNLH (SEQ ID NO: 37) F3
RASSGVSSNNLH (SEQ ID NO: 38) H1 SASSSISSSYLH (SEQ ID NO: 39) H7
SVSSSLSSSNLH (SEQ ID NO: 40) CDR- WT GTSNLAS (SEQ ID NO: 7) L2 B1
ATSSRAT (SEQ ID NO: 41) C1 GTSSRAS (SEQ ID NO: 30) C3 ATSNRES (SEQ
ID NO: 42) C10 ATSSLAT (SEQ ID NO: 43) E9, G6 GASSLAT (SEQ ID NO:
44) G3 ATSNLAS (SEQ ID NO: 45) H2 TASSRAS (SEQ ID NO: 46) CDR- WT
HGDDPAW (SEQ ID NO: 47) H3 B4, B8, C4, HGDDVAW (SEQ ID NO: 48) C6,
C10, G5 C8 HGDDIAW (SEQ ID NO: 49) CDR- WT HGDDPAW (SEQ ID NO: 47)
L3H3 A3, A8, A10, HGDDVAW (SEQ ID NO: 48) F8, H4, H6, H10 G8
HGDDISW (SEQ ID NO: 50)
Example 1-4: Fab Production Using Protein-Expressing Strain TOP10F'
and Purification
[0091] To compare the binding affinities of the selected clones for
FOLR1, purification of each clone was performed. Prior to
purification, the host strain was changed from TG1 to TOP10F' cells
to express only a Fab region.
[0092] A colony corresponding to each of the selected clones was
seeded into 4 mL SB/ampicillin medium and cultured overnight at
37.degree. C. The next day, 4 ml of the overnight culture was
seeded into 400 mL SB/ampicillin medium and cultured in an
incubator at 37.degree. C. for about 3 to 4 hours until the
OD.sub.600 reached 0.5 to 1.0. Next, for expression of the clone,
the culture was treated with IPTG to a final concentration of 1 mM
and then cultured overnight at 30.degree. C. However, when only
expression was to be confirmed, culture was performed in 20 mL of
SB/ampicillin medium.
[0093] To recover a periplasm from 400 mL of the culture, the
culture was centrifuged to remove the supernatant, and then the
cells were lysed by treatment with 16 mL of 1.times.TES solution at
4.degree. C. and incubation at 4.degree. C. for 1 hour.
Additionally, the cells were treated with 24 mL of 0.2.times.TES
solution and incubated for 1 hour. Next, the supernatant was
collected by centrifugation and 5 mM MgCl.sub.2 was added thereto
in order to remove EDTA. Before loading of the sample, a column
packed with 0.5 mL of Ni-NTA His-Bind.RTM. resin was washed with 20
CV of elution buffer (300 mM imidazole in PBS, pH 7.4) and then
flushed with 20 CV of PBS. The sample was loaded onto the column
and the flow-through was collected. After completion of loading,
the column was flushed with 20 CV of wash buffer (20 mM imidazole
in PBS, pH 7.4) and the washing-through was collected. Then, the
column was flushed with 10 CV of elution buffer and the eluent was
collected. 15 .mu.L of the sample collected in each step during the
purification process was loaded in each well in 12% SDS-PAGE and
electrophoresed (at 150 V for 1 hour). The band was visualized by
Coomassie blue staining.
Example 1-5: Examination of Direct Binding Pattern of Selected
Clone to FOLR1 at Different Concentrations
[0094] For the purpose of comparing the binding affinities of the
primarily selected clones with each other, ELISA was performed at
different clone concentrations in the following manner. 25 .mu.L of
FOLR1 was added to each well of a 96-well plate at a concentration
of 1 .mu.g/mL, coated on the plate at room temperature for 1 hour,
and then blocked with 180 .mu.L of 3% skim milk at room temperature
for 1 hour. During blocking, samples to be used as primary
antibodies were prepared. As primary antibodies, samples selected
by screening were used. The samples were diluted to a concentration
of 0.1 to 100 nM (0, 0.1, 0.3, 1, 3, 10, 30, and 100 nM) and
cold-stored until use. After blocking, 3% skim milk was removed,
and 25 .mu.L of the primary antibody was added to each well and
allowed to react at room temperature for 1 hour or more. After
completion of the reaction, each well was washed three times with
PBS-Tween20 (0.1%) buffer. As a secondary antibody, HA-HRP was
diluted 3,000-fold with 3% skim milk, and 25 .mu.L of the dilution
was added to each well and allowed to react at room temperature for
1 hour or more. After completion of the reaction, each well was
washed three times with PBS-Tween20 (0.1%), and 25 .mu.L of
substrate TMB was added to each well to confirm color development.
After about 5 minutes, 25 .mu.L of 1 M H.sub.2SO.sub.4 was added to
each well to stop the reaction, and the absorbance at a wavelength
of 450 nm was measured. From the measurement results, the EC
(Effector concentration) value was calculated using the GraphPad
Prism 7 program.
[0095] To measure the binding affinity of the selected clone for
FOLR1, SPR measurement was performed using a Biacore3000. Before
each sample was loaded, a CM5 sensor chip was activated with 0.1 M
NHS/0.4 M EDC, and then FOLR1 (20 .mu.g/mL in 10 mM acetate, pH
5.0) was immobilized thereon. Then, unreacted NHS was deactivated
with 1 M ethanolamine. 250 .mu.L of each sample was prepared at
concentrations of 1, 2, 5, 10, 20 and 40 nM, and association and
dissociation sensorgrams were obtained while each sample was
flushed at 30 .mu.l/min. The sensor chip was regenerated with 10 mM
glycine (pH 2.1). The obtained sensorgrams were analyzed using the
BIAevaluation software, and the KD values were calculated.
Example 1-6: Sequencing of Antibody Library
[0096] The sequences of the randomized complementarity-determining
regions of the clones selected by ELISA of the phage library were
analyzed. Using, as a template, 1 .mu.L of a culture of each clone
selected from the clones cultured in the 96-well plate, PCR
reaction was performed using pC3X-f and pC3X-b primers under the
following conditions: pre-denaturation at 94.degree. C. for 2
minutes, and then 25 cycles, each consisting of 30 sec at
94.degree. C., 30 sec at 56.degree. C. and 2 min at 72.degree. C.,
followed by elongation at 72.degree. C. for 7 min. Whether
amplification occurred was checked by 1% agarose gel
electrophoresis, and the amplified PCR product was purified with DW
using a QIAvac 96 and sequenced. The sequence of the
complementarity-determining region was analyzed using the leader
sequence.
Example 2: Construction of Modified Antibody (vAb)
[0097] The final clone pvAb was constructed in the same manner as
the antibody library construction method. The templates and primers
used are shown in Table 7 below, and information about the vAb
antibody sequence is summarized in Tables 8 to 10 below.
TABLE-US-00008 TABLE 7 PCR templates and primers for final clone
construction Clone PCR Template Primer Product pvAb 1.sup.st
pCDR-L1#C6 pC3X-f, Farl-L2-b CDR-L1 pCDR-L2#C1 Farl-L2-f, Lead-b
CDR-L2 CDR-L3H3#A3 Lead-VH, pC3X-b CDR-H3 2.sup.nd CDR-L2, CDR-H3
Farl-L2-f, pC3X-b CDR-L2H3 3.sup.rd CDR-L1, CDR- pC3X-f, pC3X-b
CDR-L1L2H3 L2H3
TABLE-US-00009 TABLE 8 Sequence comparison of complementarity-
determining region between parent antibody and modified antibody
(vAb) Amino Amino Position acid of acid CDR sequence of CDR Parent
antibody in parent after modified region CDR sequence sequence
antibody modification antibody CDR- HGDDPAWFAY (SEQ 103 P V
HGDDVAWFAY (SEQ H3 ID NO: 5) ID NO: 28) CDR- SVSSSISSNNLH 25 V A
SASSGLSSSYLH L1 (SEQ ID NO: 6) 28 S G (SEQ ID NO: 29) 29 I L 32 N S
33 N Y CDR- GTSNLAS (SEQ ID 54 N S GTSSRAS (SEQ ID L2 NO: 7) 55 L R
NO: 30)
[0098] Through combinations of the CDR sequences identified as
described above, the modified antibodies vAb1, vAb2, vAb3, vAb4 and
vAb5 were constructed. vAb1 is a modified antibody that reflects
all of CDR-H3, CDR-L1 and CDR-L2, vAb2 is a modified antibody that
reflects CDR-L1 and CDR-L2, vAb3 is a modified antibody that
reflects CDR-L1, vAb4 is a modified antibody that reflects CDR-L2,
and vAb5 is a modified antibody that reflects CDR-H3.
TABLE-US-00010 TABLE 9 Amino acid sequences of parent antibody and
modified antibody (vAb) SEQ ID Type Sequence NO HC
EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEW 1
VAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVY
FCARHGDDPAWFAYWGQGTPVTVSS LC
DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKP 2
WIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSS YPYMYTFGQGTKVEIK HC
EVQLVESGGGVVQPGRSLRLSCSASGFTFSGYGLSWVRQAPGKGLEW 31
VAMISSGGSYTYYADSVKGRFAISRDNAKNTLFLQMDSLRPEDTGVY
FCARHGDDVAWFAYWGQGTPVTVSS LC
DIQLTQSPSSLSASVGDRVTITCSASSGLSSSYLHWYQQKPGKAPKP 32
WIYGTSSRASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSS YPYMYTFGQGTKVEIK LC
DIQLTQSPSSLSASVGDRVTITCSASSGLSSSYLHWYQQKPGKAPKP 33
WIYGTSNLASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSS YPYMYTFGQGTKVEIK LC
DIQLTQSPSSLSASVGDRVTITCSVSSSISSNNLHWYQQKPGKAPKP 34
WIYGTSSRASGVPSRFSGSGSGTDYTFTISSLQPEDIATYYCQQWSS
YPYMYTFGQGTKVEIK
TABLE-US-00011 TABLE 10 Sequences of modified antibodies (vAb)
Modified antibody HC LC vAb1 SEQ ID NO: 31 SEQ ID NO: 32 vAb2 SEQ
ID NO: 1 SEQ ID NO: 32 vAb3 SEQ ID NO: 1 SEQ ID NO: 33 vAb4 SEQ ID
NO: 1 SEQ ID NO: 34 vAb5 SEQ ID NO: 31 SEQ ID NO: 2
Example 3: Expression and Purification of Modified Antibody
(vAb)
[0099] To produce the modified antibody (vAb), expiCHO cells were
transfected with a vector (pc 3.4-vAbL, pc 3.4-vAbH) containing the
gene encoding the modified antibody (vAb) protein and cultured, and
the modified antibody was purified using affinity chromatography.
An XK16 column packed with the affinity resin MabSelect SuRe.TM.
(GE Healthcare) was equilibrated by flushing with buffer A (25 mM
Tris, pH 7.0, 25 mM NaCl), and then the culture was flushed and
bound to the affinity resin, and the modified antibody (vAb)
protein was eluted with buffer B (25 mM citric acid, pH 3.5). After
completion of purification, the column was washed with 0.5 M NaOH,
and then packed with 20% ethanol and cold-stored. The pH of the
eluted sample was adjusted to 6.0 by adding a suitable amount of 1
M Tris (pH 9.0) thereto. The state of the sample was checked
through 10% SDS-PAGE. The obtained modified antibody (vAb) protein
was subjected to buffer exchange by dialysis against a buffer
containing 10 mM sodium succinate and 30 mM sucrose (pH 6.0).
Example 4: Analysis of Binding Affinity of Modified Antibody (vAb)
by ELISA Assay
[0100] For the purpose of comparing the binding affinity of the
modified antibody (vAb) produced in Example 2 with that of the
parent antibody, ELISA was performed at an antibody concentration
of 0.006 to 6.250 ng/mL in the same manner as described in Examples
1-5. As a result of the measurement, it was confirmed that the
modified antibodies vAb1 to vAb5 bound to FOLR1 with a 4.48- to
1.42-fold higher binding affinity than the parent antibody (Table
11). The measured values are obtained in different experiments for
comparison with the parent antibody.
TABLE-US-00012 TABLE 11 Results of ELISA assay EC50 Relative
Coating Sample ng/mL Potency R.sup.2 FOLR1-Fc Parent 10.99 1.00
0.997 antibody vAb1 2.45 4.48 FOLR1-Fc Parent 11.85 1.00 0.972
antibody vAb2 3.94 3.04 FOLR1-Fc Parent 3.04 1.00 0.996 antibody
vAb3 0.77 3.99 vAb4 1.59 1.94 vAb5 2.17 1.42
Example 5: Examination of Binding Affinity of Modified Antibody
(vAb) by SPR Assay
[0101] To measure the binding affinity of the modified antibody
vAb1 produced in Example 2 for FOLR1, an SPR assay was performed
using a Biacore3000, in the same manner as described in Examples
1-5. The sensorgram was analyzed using the BIAevaluation software,
and the K.sub.D value was calculated. The modified antibody vAb1
showed a K.sub.D value of 0.24 nM, which corresponds to a 4-fold
higher binding affinity than that of the parent antibody (Table
12). This value is similar to the relative ratio of the values
obtained using ELISA.
TABLE-US-00013 TABLE 12 Dissociation constants of parent antibody
and modified antibody vAb1, measured by SPR k.sub.a k.sub.d K.sub.A
K.sub.D Relative Ligand FOLR1 (1/Ms) (1/s) (1/M) (nM) Potency
Chi.sup.2 Analyte Parent antibody 4.19E+05 4.01E-04 1.05E+09 0.96 1
4.90 Modified antibody 6.12E+05 1.47E-04 4.17E+09 0.24 4 4.86
vAb1
INDUSTRIAL APPLICABILITY
[0102] The modified antibody according to the present invention has
a sequence in which the antigen-binding site originally possessed
by the parent antibody is substituted with an amino acid that
better binds to the antigen while preserving the basic structure of
the parent antibody. Thus, the modified antibody has increased
binding affinity for the antigen. In addition, the antibody or
antigen-binding fragment thereof according to the present invention
may be used for the prevention or treatment of cancer, and may also
be used for diagnosis of disease.
[0103] Although the present invention has been described in detail
with reference to specific features, it will be apparent to those
skilled in the art that this description is only of a preferred
embodiment thereof, and does not limit the scope of the present
invention. Thus, the substantial scope of the present invention
will be defined by the appended claims and equivalents thereto.
[0104] Sequence List Free Text
[0105] Electronic file is attached.
Sequence CWU 1
1
541119PRTArtificial SequenceHeavy Chain 1Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10 15Ser Leu Arg Leu Ser
Cys Ser Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30Gly Leu Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ala Met Ile
Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly
Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Phe65 70 75
80Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr Gly Val Tyr Phe Cys
85 90 95Ala Arg His Gly Asp Asp Pro Ala Trp Phe Ala Tyr Trp Gly Gln
Gly 100 105 110Thr Pro Val Thr Val Ser Ser 1152110PRTArtificial
SequenceLight Chain 2Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu
Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Val Ser
Ser Ser Ile Ser Ser Asn 20 25 30Asn Leu His Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Pro Trp 35 40 45Ile Tyr Gly Thr Ser Asn Leu Ala
Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp
Tyr Thr Phe Thr Ile Ser Ser Leu Gln65 70 75 80Pro Glu Asp Ile Ala
Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro 85 90 95Tyr Met Tyr Thr
Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 110310PRTArtificial
SequenceCDR-H1 3Gly Phe Thr Phe Ser Gly Tyr Gly Leu Ser1 5
10415PRTArtificial SequenceCDR-H2 4Met Ile Ser Ser Gly Gly Ser Tyr
Thr Tyr Tyr Ala Asp Ser Val1 5 10 15510PRTArtificial SequenceCDR-H3
5His Gly Asp Asp Pro Ala Trp Phe Ala Tyr1 5 10612PRTArtificial
SequenceCDR-L1 6Ser Val Ser Ser Ser Ile Ser Ser Asn Asn Leu His1 5
1077PRTArtificial SequenceCDR-L2 7Gly Thr Ser Asn Leu Ala Ser1
5811PRTArtificial SequenceCDR-L3 8Gln Gln Trp Ser Ser Tyr Pro Tyr
Met Tyr Thr1 5 10955DNAArtificial Sequenceprimer 9gcctctggct
tcactttcag trrttacgvt mtgarttggg tgagacaggc acctg
551021DNAArtificial Sequenceprimer 10actgaaagtg aagccagagg c
211166DNAArtificial Sequencen is a or g or c or t(22)..(23)n is a
or g or c or t(25)..(26)primer 11ggggtctatt tttgtgcaag annknnkgac
gatccagcat ggtttgmtta ctggggccaa 60gggacc 661266DNAArtificial
Sequencen is a or g or c or t(25)..(26)is a or g or c or
t(28)..(29)primer 12ggggtctatt tttgtgcaag acacnnknnk gatccagcat
ggtttgmtta ctggggccaa 60gggacc 661366DNAArtificial Sequencen is a
or g or c or t(28)..(29)n is a or g or c or t(31)..(32)primer
13ggggtctatt tttgtgcaag acacggtnnk nnkccagcat ggtttgmtta ctggggccaa
60gggacc 661466DNAArtificial Sequencen is a or g or c or
t(31)..(232)n is a or g or c or t(34)..(35)primer 14ggggtctatt
tttgtgcaag acacggtgac nnknnkgcat ggtttgmtta ctggggccaa 60gggacc
661566DNAArtificial Sequencen is a or g or c or t(34)..(35)n is a
or g or c or t(37)..(38)primer 15ggggtctatt tttgtgcaag acacggtgac
gatnnknnkt ggtttgmtta ctggggccaa 60gggacc 661666DNAArtificial
Sequencen is a or g or c or t(37)..(38)n is a or g or c or
t(40)..(41)primer 16ggggtctatt tttgtgcaag acacggtgac gatccannkn
nktttgmtta ctggggccaa 60gggacc 661721DNAArtificial Sequenceprimer
17tcttgcacaa aaatagaccc c 211875DNAArtificial Sequenceprimer
18acagagtcac catcacatgc agkgyttcct ccrgtvttag ttcaarcwat ctgmactggt
60atcagcagaa gcccg 751920DNAArtificial Sequenceprimer 19gcatgtgatg
gtgactctgt 202059DNAArtificial Sequenceprimer 20cccaaagccc
tggatctacg vtrcctctav tckggmaast ggggtgcctt caaggttca
592119DNAArtificial Sequenceprimer 21gtagatccag ggctttggg
192262DNAArtificial Sequencen is a or g or c or t(22)..(23)n is a
or g or c or t(40)..(41)primer 22gcaacttact attgccagca gnnkbmtwat
twtccaymtn nkymcacctt cggtcagggc 60ac 622321DNAArtificial
Sequenceprimer 23ctgctggcaa tagtaagttg c 212419DNAArtificial
Sequenceprimer 24gcacgacagg tttcccgac 192520DNAArtificial
Sequenceprimer 25aaccatcgat agcagcaccg 202618DNAArtificial
Sequenceprimer 26ggccatggct ggttgggc 182718DNAArtificial
Sequenceprimer 27gcccaaccag ccatggcc 182810PRTArtificial
SequenceCDR-H3 28His Gly Asp Asp Val Ala Trp Phe Ala Tyr1 5
102912PRTArtificial SequenceCDR-L1 29Ser Ala Ser Ser Gly Leu Ser
Ser Ser Tyr Leu His1 5 10307PRTArtificial SequenceCDR-L2 30Gly Thr
Ser Ser Arg Ala Ser1 531119PRTArtificial SequenceHeavy Chain 31Glu
Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg1 5 10
15Ser Leu Arg Leu Ser Cys Ser Ala Ser Gly Phe Thr Phe Ser Gly Tyr
20 25 30Gly Leu Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp
Val 35 40 45Ala Met Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Ala Asp
Ser Val 50 55 60Lys Gly Arg Phe Ala Ile Ser Arg Asp Asn Ala Lys Asn
Thr Leu Phe65 70 75 80Leu Gln Met Asp Ser Leu Arg Pro Glu Asp Thr
Gly Val Tyr Phe Cys 85 90 95Ala Arg His Gly Asp Asp Val Ala Trp Phe
Ala Tyr Trp Gly Gln Gly 100 105 110Thr Pro Val Thr Val Ser Ser
11532110PRTArtificial SequenceLight Chain 32Asp Ile Gln Leu Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr Ile
Thr Cys Ser Ala Ser Ser Gly Leu Ser Ser Ser 20 25 30Tyr Leu His Trp
Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp 35 40 45Ile Tyr Gly
Thr Ser Ser Arg Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly Ser
Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln65 70 75
80Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro
85 90 95Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 11033110PRTArtificial SequenceLight Chain 33Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Ser Ala Ser Ser Gly Leu Ser Ser Ser 20 25 30Tyr Leu His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp 35 40 45Ile Tyr
Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln65 70 75
80Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro
85 90 95Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 11034110PRTArtificial SequenceLight Chain 34Asp Ile Gln Leu Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg Val Thr
Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Asn 20 25 30Asn Leu His
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Pro Trp 35 40 45Ile Tyr
Gly Thr Ser Ser Arg Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60Gly
Ser Gly Ser Gly Thr Asp Tyr Thr Phe Thr Ile Ser Ser Leu Gln65 70 75
80Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro
85 90 95Tyr Met Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105 1103512PRTArtificial SequenceCDR-L1 35Ser Ala Ser Ser Ser Leu
Ser Ser Ser Tyr Leu His1 5 103612PRTArtificial SequenceCDR-L1 36Arg
Val Ser Ser Gly Ile Ser Ser Asn Asn Leu His1 5 103712PRTArtificial
SequenceCDR-L1 37Arg Ala Ser Ser Gly Leu Ser Ser Asn Asn Leu His1 5
103812PRTArtificial SequenceCDR-L1 38Arg Ala Ser Ser Gly Val Ser
Ser Asn Asn Leu His1 5 103912PRTArtificial SequenceCDR-L1 39Ser Ala
Ser Ser Ser Ile Ser Ser Ser Tyr Leu His1 5 104012PRTArtificial
SequenceCDR-L1 40Ser Val Ser Ser Ser Leu Ser Ser Ser Asn Leu His1 5
10417PRTArtificial SequenceCDR-L2 41Ala Thr Ser Ser Arg Ala Thr1
5427PRTArtificial SequenceCDR-L2 42Ala Thr Ser Asn Arg Glu Ser1
5437PRTArtificial SequenceCDR-L2 43Ala Thr Ser Ser Leu Ala Thr1
5447PRTArtificial SequenceCDR-L2 44Gly Ala Ser Ser Leu Ala Thr1
5457PRTArtificial SequenceCDR-L2 45Ala Thr Ser Asn Leu Ala Ser1
5467PRTArtificial SequenceCDR-L2 46Thr Ala Ser Ser Arg Ala Ser1
5477PRTArtificial SequenceCDR-H3, CDR-L3H3 47His Gly Asp Asp Pro
Ala Trp1 5487PRTArtificial SequenceCDR-H3, CDR-L3H3 48His Gly Asp
Asp Val Ala Trp1 5497PRTArtificial SequenceCDR-H3 49His Gly Asp Asp
Ile Ala Trp1 5507PRTArtificial SequenceCDR-L3H3 50His Gly Asp Asp
Ile Ser Trp1 5514PRTArtificial SequenceXaa is any amino acid
residue(2)..(2)Xaa is any amino acid residue(3)..(3)Xaa is any
amino acid residue(4)..(4)Synthetic construct 51Cys Xaa Xaa
Xaa525PRTArtificial SequenceSynthetic construct 52Cys Gln Trp Val
Ala1 5534PRTArtificial SequenceSynthetic construct 53Trp Gly Gln
Gly1544PRTArtificial SequenceSynthetic construct 54Phe Gly Gln
Gly1
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